Synthesis, accumulation and turnover of carbamoylphosphate synthetase and phosphoenolpyruvate carboxykinase in cultures of embryonic rat hepatocytes

Abstract

Glucocorticosteroid, thyroid hormones and cyclic AMP can induce the synthesis of carbamoylphosphate synthetase and phosphoenolpyruvate carboxykinase in cultures of hepatocytes as soon as these cells differentiate from the embryonic foregut. The low levels of both enzymes that can accumulate in such still protodifferentiated hepatocytes are due to low levels of enzyme synthesis. In cultures, the rate of synthesis of both enzymes increases continually in the presence of hormones, showing that maturation of the capacity for synthesis towards the postnatal, fully differentiated situation is occurring in these cells. The turnover rate of both enzymes in embryonic hepatocytes is lower in the presence of hormones than in the absence, but does not change during the culture period. In the presence of hormones the turnover rate is comparable to that found in adult rat liver in vivo. The development of the capacity to accumulate organ-specific enzymes in vitro (and hence the rate of enzyme synthesis) is found to be comparable to that in utero.During rat-liver development several hepatocyte-specific proteins become enzymatically detectable upon the completion of liver organogenesis. However, in the presence of glucocorticosteroids, triiodothyronine and cyclic AMP, the accumulation of the urea-cycle enzyme carbamoylphosphate synthetase and the gluconeogenic enzyme phosphoenolpyruvate carboxykinase can already be demonstrated in monolayer cultures of hepatocytes at the beginning of the organogenetic period, i.e. from the moment hepatocytes become recognizable [l, 21. The experiments further indicated that the capacity of these cells to accumulate hepatocytespecific enzymes is still limited [l], even when cultured in an optimized medium [2].The capacity to accumulate proteins is determined by the balance of the rate of protein synthesis and the rate of protein degradation. There is general agreement that in perinatal rat liver, rates of protein synthesis are relatively high and rates of protein degradation are relatively low compared to those in the adult liver [3 -51. In this period a rapid accumulation of many hepatocyte-specific enzymes is observed. Such reciprocal changes in the rates of protein synthesis and degradation have also been demonstrated for specific proteins, e.g. phosphoenolpyruvate carboxykinase [6], and may reflect the general mechanism for the rapid accumulation of specific protein during terminal differentiation of cells [7].In view of these observations, it seemed possible that the observed low capacity of embryonic hepatocytes to synthesize organ-specific proteins [l] reflects their still protodifferentiated state of development [8]. This hypothesis predicts an increase in the synthetic capacity of the (cultured) hepatocytes upon further development towards the differentiated state.

The initial accumulation of carbamoylphosphate synthetase in embryonic rat hepatocytes, and the cell cycle

“…The most striking result, however, is the fact that from that moment onwards, the number of cells accumulating enzyme increased simultaneously in all four cell cohorts, i.e., enzyme accumulated in hepatocytes independent of the position of the cell in the cell cycle. From the fact that we have no indication for a difference in biological half life of carbamoylphosphate synthetase protein in embryonic hepatocytes when compared to adult hepatocytes [29], we conclude that enzyme accumulation reflects enzyme synthesis. Figure 3D, in which ['HI-TdR was used as the first and bromodeoxyuridine as the second label, shows that the carbamoylphosphate synthetase accumulation is retarded in the purely bromodeoxyuridine-labeled cell cohort only, i.e., in cells exposed to bromodeoxyuridine at the beginning of the S phase.…”

The expression of liver-specific genes within rat embryonic hepatocytes is a discontinuous process

“…We have presently extended these results, showing that when steady-state conditions are reached after induction, i.e. when the enzyme content per average hepatocyte does not change anymore [22,23], the degree of intercelluar heterogeneity is higher for short-lived proteins (such as PEPCK [36]) than for long-lived proteins (such as CPS [36]) and higher when suboptimal (tenfold lower) concentrations of inducing hormones are present than when optimal concentrations are provided. Likewise, the degree of interhepatocyte heterogeneity that persists under steady-state induced conditions [37] is higher for mRNAs than for their respective proteins and higher for PEPCK mRNA than for CPS mRNA.…”

“…period [22,37,381, thus indicating regulation at both the transcriptional and translational level. Although naive embryonic rat hepatocytes can be induced to accumulate liver-specific proteins, their synthetic capacity is low [36]. We have observed that maturation towards the adult synthetic capacity in these hepatocytes in our in vitro culture system closely mimics hepatocyte maturation in vivo [23, 251. Immunostaining of cultured hepatocytes with antibodies directed against CPS and PEPCK has shown that the initiation of expression of both genes is a stochastic event [38].…”